water flow and gas exchange at the gills of...

J. Exp. Biol. (1971). 54. 1-18 IWith 6 text-figuret

Printed in Great Britain

WATER FLOW AND GAS EXCHANGE AT THE GILLS OFRAINBOW TROUT, SALMO GAIRDNERI

BY JOHN C. DAVIS AND JAMES N. CAMERON

Department of Zoology, University of British Columbia,Vancouver 8, B.C., Canada

(Received 10 August 1970)

INTRODUCTION

The Fick principle has often been used to estimate the volume flow of water perunit time over the gills of fishes. Application of the Fick principle requires accuratemeasurement of mean oxygen tensions in water entering and leaving the gills and oftotal oxygen uptake. Cannulae of various types have frequently been used to samplewater entering and leaving the gills (van Dam, 1938; Holeton & Randall, 1967a, b;Randall, Holeton & Stevens, 1967; Saunders, 1961,1962; Stevens & Randall, 1967 a, b).Recent studies have indicated that these cannulation techniques sometimes providepoor estimates of mean expired oxygen tensions (Davis & Watters, 1970; Garey, 1967).Procedures of this type therefore could lead to inaccurate estimates of volume flowof water over the gills. Hence it was necessary to devise another method of measuringventilation volume in this study.

Variations in ventilation volume influence gas exchange across the gills. Hughes(1966) and Saunders (1961) discussed the effects of high gill water flow, pointing outthat anatomical deadspace increases under these conditions since portions of theventilatory flow are shunted past the tips of adjacent gill filaments. It was a furtherobjective of this study to see how the velocity of water flow over the gills influenceda fish's ability to exchange gases with the water.

METHODS

Explanation of symbols used

VQ, volume of water passing over the gills/unit time (ventilation volume)VR, ventilation rate in mouth or opercular closures/minuteVsv, volume of water pumped over the gills/breathing cycle (ventilatory stroke

volume)% U, percentage of oxygen utilized from inspired waterVOt, oxygen uptake/unit timeP, partial pressure of gas in mmHga, solubility coefficient for gas in water or bloodQ, cardiac output/unit timeS.D., standard deviationS.E., standard error of meanHct, packed cell volume of blood in vol. % (haematocrit)

I BXBJ4

J. C. DAVIS AND J. N. CAMERON

Explanation of symbols used (cont.)B, referring to bloodW, referring to water

SubscriptsO2, COa, molecular speciesa, arterialv, venous/, inspiredE, expired.

Experiments were carried out during 1969 at the Vancouver Public Aquarium,Vancouver, B.C. Stocks of rainbow trout, Salmo gairdneri, were purchased from theSun Valley Trout Farm, Coquitlam, B.C., and were held in 250 gallon tanks prior touse. The fish were fed with a mixed diet of Clark's trout pellets and chopped horseheart twice weekly.

Test fish were anaesthetized in a bucket containing 1:10000 MS 222 solution andwere transferred to an operating table similar to that described by Smith & Bell (1964).This device was equipped with a pump and nozzles to perfuse the gills with anaestheticsolutions during operations.

Retaining Oral membranering \ /

Holding tube

1Water level

"\'"t ' • • " " ' " " " " - ' • • • • ' • • • • • " " " " " ' " " • • • •'••'""'*r"M'"""'i"*"rrmr""M

Overflow

Fig. 1. The apparatus for direct measurement of ventilation volume in trout. The fish has thethumb-portion of a rubber surgeon's glove stitched round the margin of its mouth. The rubbermembrane separates inspired and expired water and allows collection of the latter.

Direct measurement of ventilation volume

A series of experiments was conducted to measure directly the volume of waterflowing over the gills of trout/unit time. The technique involved attachment of aportion of a thin rubber surgeon's glove to the mouth of a trout so that it did notinterfere with breathing but allowed direct measurement of ventilation volume byseparating inspired and expired water (Fig. 1).

A dorsal aortic cannula was implanted in the fish as described by Smith & Bell(1964) and a 40 cm length of number o silk was tied through the anterior portion of

Gill water flow in trout 3

the upper jaw as a restraint upon the fish. Next, a 12 cm circle which included thethumb was cut from a number 8£ latex surgeon's glove. The tip of the thumb wascut to fit the fish's head so that it covered the dorsal aortic cannula at its point ofemergence but left the eyes uncovered. The membrane was then stitched around themargin of the fish's mouth with number 000 silk sutures placed 2-3 mm apart. Whenproperly cut and positioned the membrane exerted little tension on the head or jawsand had sufficient slack to permit easy movement of the branchial apparatus. On twoof the subjects the membrane was too tight and the fish were unable to open and closetheir mouths properly. Results from these two fish were discarded.

Fish with attached oral membranes were placed in a Lucite box divided into twochambers by a partition with a hole through it. The membrane was attached to theedge of the hole by a Lucite retaining ring and brass wingnuts. Thus the membraneserved as a barrier between the two chambers and separated inspired and expiredwater. A rectangular Lucite box, open at both ends, supported the fish while its headwas anchored to the retaining ring by the string through the upper jaw. Movementwas further restricted by a Lucite bar across the retaining ring so that the fish couldnot swim forward and tear loose from the membrane. When in place the trout couldmove its head freely about 2 cm in any one direction but could not exert a direct pullon the membrane. The top and sides of the box were covered with black plastic toprevent the fish seeing the investigators.

The drains at either end of the box could be set at various levels by sliding themup and down through rubber stoppers. Following the operation the drain at the headend of the box was raised to provide positive pressure in the buccal cavity and facilitateventilation during recovery. At that time the membrane could be checked for leakagewith dye. When the membrane was properly affixed very little leakage was apparentwith a pressure differential of 2-3 cm H2O across the membrane.

The trout were allowed overnight recovery following attachment of the membrane.Then the drains in the box were levelled and the fish was left undisturbed for at least1 h. Ventilation rate (VR), ventilation volume (Po), dorsal aortic oxygen tension(Pa(o,))> inspired oxygen tension (Pnoj), and expired oxygen tension (P#(o,)) werethen measured at intervals. Ventilation volume was measured by collecting the waterspilling over the rear drain as the fish breathed. Water was collected for 1 min andventilation rate was determined simultaneously by counting the number of mouth oropercular movements. Often these measurements were carried out on the same indi-vidual for a period of 2 or 3 days. In a few cases oxygen-deficient water (56-65 mmPo%) was run into the box and the fish's response was studied.

Oxygen tensions in blood and water were measured with a Radiometer electrodesystem as described by Cameron & Davis (1970) and Davis & Watters (1970). Oxygenuptake was calculated by the Fick principle using the inspired oxygen tension, theexpired oxygen tension from the rectangular body-holding chamber, the measuredventilation volume and the oxygen solubility coefficient in water at the test temperature.Cardiac output was calculated from the oxygen uptake, the dorsal aortic PO i and anassumed venous PO i of 32 mmHg. We believe this assumption to be justified as thesecond portion of this study, the results of Cameron & Davis (1970) and those ofHoleton & Randall (19676) all indicate that P^o,) m trout remains steady at 30-35 mmHg. Blood oxygen content calculations were based upon unpublished blood

4 J. C. DAVIS AND J. N. CAMERON

curves for rainbow trout by Beaumont, Holeton & Randall and the relationshipbetween haematocrit and blood oxygen content determined by Holeton & Randall

(1967*)<xBOt = 0-311 Hct + o-y,

(where aBOi and Hct are in volumes %). Measurements of ocB0t in rainbow troutusing Tucker's (1967) technique agree with those predicted by Holeton & Randall'sformula. It was assumed that venous + arterial blood contained 2-5 and 1-1-5 m m

^co, respectively (Holeton & Randall, 19676).

Gill per fusion studies

To study the effect of varying water flow over the gills it was necessary to devise amethod of artificially perfusing the gills of intact trout at different flow rates. Dorsaland ventral aortic cannulae were implanted (Smith & Bell, 1964; Holeton & Randall,1967a) and a 4 cm length of stiff Tygon tubing (9 mm I.D., 12 mm o.D.) was tied intothe mouth. The tube was tied securely so that it penetrated about 1 cm into the buccalcavity and directed a flow of water toward the gills in a plane parallel with the roofof the mouth. The mouth tube eliminated any activity of the buccal pump but theopercular apparatus was free to move.

Following cannulation the trout was placed in a Lucite box similar to the constant-flow system described by Fry (1957). Lucite pegs inserted into holes in a baseplatewithin the box held the fish upright and kept its body straight. This form of restraintprevented the fish turning its head so that more water could flow over the gills on oneside of the body than the other. The top of the box was secured with a gasket andwingnuts and cannulae passed out of the box through needles inserted in rubberstoppers (Fig. 2). Black plastic covered the top and sides of the box.

Mouthperfusion

tube

11 ^{/- ^ \Chamberoutflow

Flow-ratemeasurement

Fig. 2. The apparatus for artificial perfusion of trout gills at different water flow rates. A tubeis tied into the mouth and the fish is supported with Lucite pegs. Cannulae for blood andwater sampling are illustrated.

Oxygen tension in inflow water, outflow water and dorsal and ventral aortic bloodwere measured with a Radiometer electrode system. All attempted measurements ofP c o in water and blood with this system were unsuccessful as PC O i was below the3 mrnHg limit of the scale. Blood samples were returned to the animal by backflushingthe cuvettes with heparinized Cortland saline (Wolf, 1963). Pressures in the variouscannulae were monitored with Statham model P 23 BB pressure transducers and aBeckman RS Dynograph recorder.


The fish were allowed to recover for 24 h following cannulation since these pro-cedures involve considerable trauma (Houston, DeWilde & Madden, 1969). Followingrecovery the gills were perfused with water for a period of 1 h at a given rate. At theend of that hour POt and blood pressure were recorded from the appropriate cannulaeand the flow was reset to a new level. Flows ranged from 45 to 1200 ml/min and wereapplied at random with the exception that the lowest flows were not used until theend of the experiment. In these experiments 41 trout ranging from 195 to 388 g werestudied at temperatures of 10-13-5 °C-

RESULTS

Direct measurement of ventilation volume

Mean ventilation volumes and other parameters from 18 rainbow trout are sum-marized in Table 1. The number of observations used for each mean Vo, VR or Vsvis given in column 3 of the table.

Table 1. Circulatory and ventilatory parameters for 18 trout (210-3 ± 2-3 g) at 8-6 °Cwith oral membranes attached for direct determination of ventilation volume

(All the values are means with the exception of those for minimum and maximum VQ whichare individual observations for each fish.)

Fishno.

i

2

3456789

IO

I I

12

•3•41516

1718

Av.nS.D.S.B.

Av. 1>G±S.E.

(ml/min)

48-338-429-131-426-034749-033741-144-635'33 1 1

2 7 1

4 0 2

49°29-137-6396

3 7 018

7 4i-8

2-7

3'33'02 - 0

0-92-42 31 9i-64-0o-62-4i-82-82-3'•52 ' 2

!-5

n

965794656

11

56

1 0

1 0

4756

Av. VR(no./min)

98-794'768-86 7 06836i-o

108277°86-562-473-86 7 859'i6i- 7

6 8 059-°71-275 0

73-818

H-33'4

Vtv(ml)

o-5°0 3 90-410-470-34o-s70-460-440 4 80 7 1

0-480-470-460-650 4 80-50°53°-53

0 4 9180-090-02

Min.VG

(ml/min)

37'O30-02 7 02 8 0

24-029-041-027-536-040-034-°2 4 022-032-O27-O2 0 0

33°35°3 0 7185-61-3

Max.x?G

(ml/min)

162-01440147046-027-0

1180

9 4 °840

129-01380

38043-°40-057-°5 9 °

114-076-071-0

88218

43'71 0 3

Av. %U

(ml/min)

3 9 744-85°-847'949-551-328-55°'348-039°41-048948-737946549-65°-855'44 6 018

6-51-5

0(ml/min)———3-7—6-o4-1

—

6-53-22-7

2-3—2-63 °2-83 65-6

3 912

1-40-4

VGI

0———8-5—5-8

I2-O

6 41 4 0

1 3 3io-8—

15-5I I - I

10-4io-6

7 - 0

10-412

3 10 9

?o(mg/kg/

h)

64857349-352-444-26i-o47-i59°6 8 350-25°-547-841-453-85 3 °54-46377 5 9

55-3188 92-1

Mean ventilation volume for the group, weight 2io±3-og, at 8-6±o-2°C was37+ i-8 ml/min when the animals were quiet. In one individual Va fell as low as22 ml/min and in another rose as high as 162 ml/min. VQ rose sharply when the fishstruggled or was disturbed by tapping on the box (Fig. 3).


Ventilatory stroke volume was normally about 0-5 ml/breath and increased markedlyas ventilation volume went up. During struggling or when the animal was disturbedin some way Vsv was high.

Ventilation rate usually varied little for any one individual despite the fact thatobservations were often made on the same individual for 2 consecutive days. Therewas considerable variance in VR between individuals however, and those with highmean T Gs tended to have high mean VRs (cf. Tables 1, 2).

Fig. 3. Typical ventilation volumes recorded from a quiescent trout over a 7 h period. Onlythose points underlined were used to determine resting mean VQ for this fish. The effect ofdisturbing the fish by tapping on the box is shown.

Utilization of oxygen from the water passing over the gills, calculated from

had a mean value of 46-1 ± 1-5 % and ranged from 28-5 to 64%. Utilization decreasedas T o increased when the fish was disturbed or struggled. The oxygen content ofinspired water was virtually constant for any one experiment and ranged between157 and 163 mm PO j over the entire study.

Arterial Po% was quite variable, ranging between 82 and i3ommHg. This rangeof tensions represents a small variation in blood oxygen content. Unpublished dis-sociation curves for rainbow trout blood (Beaumont, Holeton & Randall) show it tobe 85-100% saturated with oxygen over that POi range.

Calculated cardiac output values for the trout ranged between 2-6 and 6-5 ml/min/fish and had a mean of 3-9! 0-4 ml/min/fish. These cardiac outputs, together withthe appropriate ventilation volume values for each animal, were used to determineventilation-perfusion ratios, the ratio of water flow to blood flow over the gills. Ven-tilation-perfusion ratio (VQIQ) had a mean of 10-4 and ranged from 5-8 to 15-5.

Oxygen uptake levels for quiescent trout ranged between 41-4 and 75-9 mg/kg/hand had a mean of 55-3 ±2-1 mg/kg/h (Table 1).

Simple correlation coefficients between the various circulatory and ventilatoryparameters for quiescent fish are given in Table 2. A significant correlation (o-oi level)is shown between % U and Va. There is also a positive correlation between Vsv andweight (0-05 level).


Ventilation volumes and other parameters for four fish subjected to hypoxia aresummarized in Table 3. These data were recorded no later than 5 min after the oxygentension of the inspired water was reduced rapidly to approximately 60 mm POj.During hypoxia T j rose as high as 276 ml/min in one individual and ventilatory strokevolume went up to 3-7 ml/breath. Averaging the data for the four fish indicates thatventilation volume increased nearly sevenfold in response to hypoxia. Ventilation raterose only slightly from the level accompanying normoxic conditions. Thus the increasein ventilation volume during hypoxia was largely a result of increased ventilatorystroke volume rather than a large increase in ventilatory rate.

Table 2. A simple correlation analysis on circulation and ventilation data using themean values for 18 trout with attached oral membranes

(Only correlations which were significant at the 0-05 level or better are given.)

VG VR Vtv % U Pb, 0 VolQ Hct Weight Temp.

VRVtv

%u

0VQIQ

HctWeightTemp.

o-68»»0-46*o-67*#

o-47*N.S.N.S.N.S.N.S.N.S.

N.S.0-51*N.S.N.S.N.S.N.S.N.S.0-49*

——

N.S.N.S.N.S.N.S.N.S.O-52*N.S.

—

N.S.N.S.0 -69"N.S.N.S.N.S.

—

—

O - 7 3 "0-62*N.S.N.S.N.S.

—

0-84"o-76#<

N.S.N.S.

——

—

———

' 0-63*N.S.N.S.

—

—

N.S.N.S. N.S.

• = Significant at 005 level. • • = Significant at o-oi level. N.s. = Not significant at 0.05 level.

Table 3. Ventilatory changes in four trout that resulted when the inspired oxygen tensionfell rapidly to approximately 60 ntmHg

(The values during hypoxia were determined within 5 min of introducing the hypoxic water.)

Before hypoxia During hypoxia

Av. VQ± AV.VR± VQ VRIncrease

Weight Temp. S.E. s.E. Vtv (ml/ (no./ Vtv VR Vtv(g) (°C) (ml/min) (no./min) (ml) min) min) (ml) (no. of times)

330-0 78 35-8 34 58-8 4-9 o-6i 190-0 73 264 1-22 4-33232-0 7-8 27-5 0-7 72-0 o-8 0-38 276-0 74 3-73 1-03 9-82182-3 7'5 36-4 1'4 7O-4 i'7 0-52 I94'O 96 203 1-36 3-88238-6 7-1 24-4 0-5 72-0 o-6 0-34 1850 76 2-43 1-06 7-15

Av.220-8 7'6 3I-O 68-3 0 4 6 2II-3 79-5 2-71 I-I7 6-30

Gill per fusion experiments

Inspired oxygen tensions measured in the inflow water to the chamber were fairlyconsistent over the entire flow range studied, ranging from 153 to 162 mmHg. Oxygentensions in outflow water from the box reached a low of 85 mm P o at the minimumflow rate applied (45 ml/min) and went up as perfusion rate increased, reaching amaximum value of 154 mm at the highest perfusion rate (Fig. 4). Percentage utilizationof oxygen from the water, calculated from the above data, dropped from 44% at the

8 J. C. DAVIS AND J. N . CAMERON

lowest perfusion rate to a minimum value of 3-8% as the perfusion rate increased(Fig. 4)-

Mean dorsal aortic PO j ranged from 131 to 135 mmHg at gill water flows above500 ml/min and diminished at lower perfusion rates, reaching 44 mm Fn(0 > at a per-fusion rate of 45 ml/min (Fig. 5). Ventral aortic POi remained virtually unchangedover the entire flow range at 31-61 o-6 mmHg.

160 -

140

120

oa.

100

80

40

£=30co•£ 20

• •Box Inflow H,O*» Box outflow HaO

200 400 600 800 1000

200 400 600

Perfusion rate (ml/mln)

800 1000

Fig. 4. Oxygen tensions in water entering and leaving the box and the percentage utilizationof oxygen from the inspired water for the group of perfused fish. The data are for 41 fish, meanweight 261 ±7-6 g, at 10—135 °C. The oxygen tensions are means ±2 standard errors andutilizations are calculated from the data in the upper graph.

Blood pressure data was converted to area mean pressure according to the formula

area mean pressure =systolic pressure + 2 diastolic pressure

Area mean pressure gives a good approximation of average pressures that occur invessels during pulsatile flow (Burton, 1966). Area mean blood pressure in the ventralaorta remained steady at around 35 mmHg over the entire flow range. Dorsal aorticpressure averaged 29*3! 1-9 mmHg at a perfusion rate of 80 ml/min and droppedslightly as perfusion rate increased, reaching a low of 21-8 ± 2-0 mmHg at a flow of1043 ml/min. A Me9t showed these two values were significantly different at the0-05 level.

Gill water flow in trout g

Oxygen uptake rate, calculated by the Fick principle, rose from 56-2 to 104-9 m g /kg/h as the flow rose to 750 ml/min (Fig. 5). At higher gill water flows oxygen uptakeremained around 100 mg/kg/h. Cardiac output, calculated from the preceding data,rose in accordance with the increase in perfusion rate. At a flow of 85 ml/min cardiac

E100

200 400 600 800 1000

-aE

. . E

3 2

O ^ISO

40

30

90

60

30

0

•100

70

40

| 100

I SOB,

O 0

• ^ 80

60

200 800 1000400 600

Perfiwion rate (ml/mln)

Fig. 5. Circulatory and ventilatory parameters recorded from 41 trout whose gills were perfusedat different flow rates. Vot and Q were calculated using the means for the group. Other valuesare means ± 2 standard errors.

output was approximately 4 ml/min/fish while at a flow of 750 ml/min Q was 6-5 ml/min/fish. At perfusion rates above 85 ml/min heart rate showed little change, remainingaround a mean value of 6 3 ! 3*4 beats/min. At the lowest flow tested (45 ml/min)cardiac output, calculated by the Fick principle using an assumed P^o%) of 34 mmHgwas 15-4 ml/min/fish. This Q value is considerably higher than those at greater per-fusion rates. In addition, heart rate at the minimum perfusion rate was 49 beats/min—a rate somewhat lower than at any other perfusion rate.

io J. C. DAVIS AND J. N . CAMERON

The above data were used to calculate the transfer factor for oxygen across thegills. Transfer factor (To,) is a measure of the relative ability of the respiratory surfaceto exchange gases and may be calculated by dividing the oxygen uptake rate by thepressure gradient for oxygen across the gills (Randall et al. 1967)

Transfer factor was 0-0078 ml 02/mLn/mmHg at the lowest flow rate and increasedby about 50% as the flow rose to its highest level (Fig. 6).

^002E

2-0-01

000

40

30

I t 20

3

800 10000 200 400 600Perfuslon rate (ml/min)

Fig. 6. Area mean dorsal aortic pressure (means ± 2 standard errors) and the transfer factorfor oxygen across the gills of perfused fish.

Opercular closure rate was zero at perfusion rates in excess of 580 ml/min butincreased rapidly as flow dropped below this level, reaching a maximum rate of 85closures/min at a perfusion rate of 95 ml/min (Fig. 5).

Pco levels in both water and blood afferent and efferent to the gills were alwaysbelow 3mmHg and could therefore not be measured.

DISCUSSION

Measurement of ventilation volume

Basically, two techniques have been used to determine ventilation volume in fish:(1) the direct technique where all the expired water is collected and (2) indirectmethods where ~7a is calculated by the Fick principle or is estimated by dye dilution.A summary of literature values for Vo in teleosts and the methods used to obtain thesevalues is given in Table 4.

Recent experiments have indicated that expired oxygen tensions recorded fromopercular cannulae in trout (Davis & Watters, 1970) or carp (Garey, 1967) are highlyvariable. It was felt that this variability could lead to inaccurate estimation of meanexpired oxygen tension. Mean expired oxygen tension has frequently been used tocalculate ventilation volume by the Fick principle. Owing to these limitations a directmethod of T o measurement was adopted in this study.

Tab

le 4

. V

enti

lati

on v

olum

es a

nd o

ther

par

amet

ers

mea

sure

d or

est

imat

ed fo

r te

leos

ts u

nder

a v

arie

ty

of c

ondi

tion

s

(The

met

hods

use

d to

est

imat

e V

g ar

e: i-

dir.

col

l. =

dir

ect c

olle

ctio

n of

exp

ired

wat

er w

ith r

ubbe

r sa

cs o

n th

e gi

ll ou

tflo

w, 2

-Fic

k =

cal

cula

ted

by th

eFi

ck p

rinc

iple

fro

m V

o, a

nd th

e ox

ygen

pre

ssur

e gr

adie

nt a

cros

s th

e gi

lls, 3

-van

Dam

= a

rub

ber

van

Dam

-typ

e m

embr

ane

stre

tche

d ov

er t

he h

ead,

4-ca

lc.

= c

alcu

late

d fr

om th

eore

tical

con

side

ratio

ns, 5

-ora

l mem

. =

the

ora

l mem

bran

e of

the

pres

ent

stud

y.)

Spec

ies

Sphe

roid

ei m

acul

atus

Cat

osto

mui

com

mer

som

Icta

luru

s ne

bulo

tus

Cyp

rint

u ca

rpio

C.

carp

ioC

allio

nyrm

u ly

raM

ugil

ceph

alut

Icta

luru

s pu

ncta

tus

Tin

ea t

inea

T.

tinea

Anq

uilla

vul

gari

sK

atsu

wom

u pe

lam

iiM

acke

ral

Salm

o ga

nrdn

eri

S. g

atrd

neri

S. g

aird

neri

S. th

asta

Ref

eren

ce

1 2 2 3 2 4 S 6 7 8 91

0

11 12

13

14 9

Tem

p.(°

C)

12-2

22

0

20 10

20

11-1

213-2

12

3-2

417-2

019-2

016-1

8— — 0

-19

5 910-1

2

Wt

(kg)

02

15

01

81

2-6

01

74

00

7-0

-14

0-0

8-0

-29

00

07

00

5-0

-07

0-0

7-0

-13

0-4

1-0

-92

16

7—

O-2

I-I-

I0

-2-0

40'2

IO

'OO

0,

upta

ke(m

g/kg

/h)

17-1

03

90

7i

28

10

0 651

20

20

6—

63-1

40

57-7

74

19 5i

100-1

35

26-1

31

55 67

%u

(ml/k

g/m

in)

45-4

82-

688-

78 73 5-85 40 66 47 — 67

65-8

0— 53 55 1

0 46 80

0(m

l/kg/

min

)

— — 18 — — — — — — — — 12

0

65-1

00

6-27 3

9—

(ml/k

g/m

in)

— — 12

— — — — — — — — 152-

7-55

95-1

06

10

—

(ml/k

g/m

in)

17-1

17*

391-8

063

23

9-2

70

92

14

331-2

891

30

*

2O

-IO

I*9

25

39

-16

4*

2O-5

O-J

-9

0-1

53

2014

1830

27

4-3

56

0571-2

855

37*

148

Met

hod

of V

Gde

term

inat

ion

i-di

r. c

olL

2-F

ick

2-F

ick

2-F

ick

2-F

ick

i-di

r. c

oll.

3-va

n D

am3-

van

Dam

3-va

n D

am1,

33-

van

Dam

4-ca

lc4-

calc

2-F

ick

2-F

ick

5-or

al m

em.

3-va

n D

am

Ref

eren

ces:

i-H

all

(193

1); 2

-Sau

nder

s (1

962)

; 3-

Gar

ey (

1967

); 4

-Hug

hes&

Um

ezaw

a (1

968)

; 5-

Cec

h (1

970)

; 6-G

eral

d &

Cec

h (1

970)

; 7—

Hug

hes

&Sh

elto

n (1

958)

; 8-

Schu

man

n &

Piip

er (

1966

); o

-van

Dam

(193

8);

10-B

row

n &

Mui

r (1

969)

; 11

-Rah

n (1

966)

; 12

-Hol

eton

& R

anda

ll (1

9676

); 1

3-

Stev

ens

& R

anda

ll (1

9676

); 1

4-pr

esen

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Most estimates of Vo in Table 4 are in ml/kg/min although the studies were notmade on 1 kg fish. There is no evidence that VQ is directly related to weight so con-version of Vo estimates to ml/kg/min may introduce an additional source of error.Indeed, metabolic rate and gill surface area increase with body weight to the powerof about 0-8-0-9 (Muir, 1969; Muir & Hughes, 1969; Price, 1931) and the growth ofother parts of the body is allometric (Prosser & Brown, 1962). Mouth size in fish, andconsequently ventilation volume, probably follow a similar pattern.

Table 4 reveals considerable differences among ventilation volume estimates madewithin and between different species of teleosts. These estimates were made over awide range of experimental conditions (fish size, temperature, degree of activity,varying oxygen tensions, varying carbon dioxide tensions) and are hence not directlycomparable without consideration of these conditions.

Estimates of ventilation volume for rainbow trout (Holeton & Randall, 1967) obtainedby indirect means (opercular cannulation) range from 274 to 3560 ml/kg/min. Holeton& Randall's maximum Va is for a group of trout under severe hypoxia for a long period(35~4° rnin 1*0, after l~2 h progressive hypoxia). Stevens & Randall (1967) reportedthat Vo ranged from 571 to 2855 ml/kg/min for 200-400 g trout at 5 °C in a tunnel-type respirometer with water flowing past the fish. This range represents indirectestimates of Va for quiescent to moderately active trout in the respirometer. In thepresent study the Vo of quiescent trout was 3 7 ! i-8 ml/min (171 ml/kg/min), a valuelower than the minimum values reported above. The difference may be related tovariations in experimental conditions or in part to the techniques used to determineJ^j. Van Dam (1938), using a direct technique of measurement on a single 900 g trout,obtained Vo values very similar to those of the present study on a per kg basis.

Oral membranes could produce inaccurate measures of Vo if they caused impairmentof ventilation. No such impairment was detected as the dorsal aortic blood remained85-100% saturated and oxygen uptake values were consistent with those measuredfor similar-sized rainbow trout at 4-8 °C (Stevens & Randall, 19676). Indeed, themembrane was designed so that the tension necessary to produce a seal was exertedlaterally from the snout to the corner of the mouth along the maxilla, and from thecorner of the mouth to the tip of the mandible. There was virtually no tension exertedon the mouth in a dorso-ventral direction. In addition, fish with oral membranes werecapable of a sevenfold increase in Vo in response to hypoxia (Table 3). Since the fishwere able to increase Vo to this extent it seems clear that the membrane did notsignificantly restrict pumping. Tests with dye showed that leakage was not a significantsource of error.

In over 200 separate measurements of percentage utilization from fish with oralmembranes percentage utilization never rose as high as 80% as reported by van Dam(1938). Utilization ranged from 23 to 64% and was usually around 50%. It is possiblethat the tight rubber membrane stretched over the head of van Dam's fish impairedpumping to some degree. Preliminary experiments in our laboratory indicated thatrubber or plastic collecting bags attached to the operculae of trout produced low T^sand high utilizations compared to fish with oral membranes. In some cases the restric-tion was severe enough to kill the fish. Van Dam's trout was large (900 g) and it ishard to say if a serious restriction of breathing existed in a fish of that size. Presumably,the restriction would be greater in small fish owing to the sensitivity of the operculae


to physical loading and the increased relative tension (Laplace's law). Furthermore,van Dam's Va estimates compare closely with ours on a per kg basis. Indeed, the 80 %utilization reported by van Dam may be related to the large size of his fish. G. F.Holeton (personal communication) observed that larger trout tend to have highutilizations. Table 2, however, shows no correlation between weight and % U, possiblybecause too small a size range of fish was examined.

Oral membranes then, appear to be useful for measurement of Vo and other para-meters in quiescent trout providing restraint of the fish can be tolerated. They do notappear to produce any great restriction of breathing in 200 g fish. Oral membranesallow repeated and accurate determination of oxygen utilization and ventilationvolume. Fick principle calculations of ventilation volume depend upon accuratedetermination of mean expired oxygen tension. Opercular catheterization, a techniqueused to determine mean P#(o,)> often produces highly variable results and could leadto errors in determining mean Pgxoj)- Oral membranes are free of such limitations asthey allow direct T j measurement and provide a mixed sample of all the expired waterfor determination of mean P^o,)- The authors suggest that the reliability of meanPsAO ) measurements from cannulae should always be checked using an independentmethod.

The ventilation-perfusion ratio for trout in this study was 10-4! 0-9. Holeton &Randall (19676) reported that VajQ ranged from 2-7 to 55 (normoxic to severe,prolonged hypoxia) and Stevens & Randall (1967 A) reported a VojQ range of 95-106for trout subjected to a range of swimming activity in a respirometer. The large variationin ventilation-perfusion ratio reported for trout in these three studies could haveresulted from the different conditions present in each experiment and/or the techniquesused to determine VQIQ. In each case Q was determined by the Fick principle, aprocedure which requires accurate knowledge of tensions of oxygen and carbondioxide in arterial and venous blood and a representative oxygen dissociation curve forthe blood. If the blood curve was slightly in error or the magnitude of the Bohr shiftwas not correctly estimated then inaccurate values for Q would result. In the presentstudy it was not possible to measure blood PQO,' Thus our calculated Q depends uponthe assumption that venous and arterial blood of quiescent trout contained 2-5 and1—1-5 mm PCOj respectively as reported by Holeton& Randall (19676). VQ/Q estimatesbased on theoretical considerations (Rahn, 1966) or determined for other fish species(Baumgarten-Schumann & Piiper, 1968; Hanson & Johansen, 1970; Piiper &Schumann, 1967; Robin, Murdaugh & Millen, 1966; Garey, 1967) range from 8to 20.

Although there was only a small increase in ventilation rate during hypoxia, Table 2shows Va strongly correlated with VR. This is because individuals showed very littlevariation in ventilation rate but some had higher rates than others. Those individualswith high rate also had high Va, hence the positive correlation between these twoparameters.

Gill per fusion studies

The velocity and pattern of water flow over the gills must have a considerable effecton gas exchange. If the mouth tube directed water over the gills in a pattern of flowthat was not optimum for gas exchange then impairment of exchange might result.


Perfused fish were unable to saturate their arterial blood with oxygen at the lowestflow rate tested (Fig. 5). This perfusion rate (45 ml/min) approximates the Vo ofquiescent trout with oral membranes attached. Thus it would appear that artificialperfusion of the gills by means of the mouth tube provides less effective conditionsfor gas exchange than those present during normal ventilation.

It is likely that the restriction of exchange resulted from a poor pattern of waterflow over the gills during perfusion. Probably only a portion of the gill sieve receivedadequate ventilation at the lowest perfusion rate. A flared perfusion tube might havedirected the water flow more uniformly over the gills and resulted in a more completesaturation of the blood at the lowest flow. At high perfusion rates the dorsal aorticblood was saturated but utilization was reduced as a result of the high flow rates.

Utilization at the lowest perfusion rate was 44%, a value nearly identical with themean utilization (46%) of trout with oral membranes at a mean Vo of 37 ml/min.Thus although the dorsal aortic blood was not saturated with oxygen at this lowperfusion rate some compensation must have taken place within the fish to maintaina normal utilization when a poor pattern of flows existed at the gills. Fig. 5 showsthat cardiac output was high at the lowest perfusion rate in comparison to Q at higherperfusion rates. This high Q value was calculated by the Fick principle using anassumed P^o,) of 34 mmHg and may therefore be subject to error. An increase in Qin response to diminished Pa(oj would facilitate utilization and maintain an adequateJ^Oi despite the poor flow conditions at the gills. Rainbow trout increase their cardiacoutput in response to hypoxia (Holeton & Randall, 19676) or anaemia (Cameron &Davis, 1970) when environmental oxygen is low or the oxygen carrying capacity of theblood is reduced.

Opercular movements appeared at perfusion rates below 700 ml/min and increasedin frequency as flow rate declined (Fig. 5). There are two possible sources for initiationof this opercular activity. Ventilation could be controlled by information from receptorsites on the gills sensitive to changes in water flow. Sutterlin & Saunders (1969) describedproprioceptors associated with gill filaments and rakers of the sea raven and Atlanticsalmon. These receptors responded to mechanical displacement. Alternately, theopercular activity could be related to arterial oxygen tension. Opercular movementwas initiated at roughly the same flow rate as that when dorsal aortic Po began todecline (Fig. 5). It is possible that trout possess a receptor system capable of adjustingventilation in response to changes in arterial Po . The pseudobranch could be the siteof such a system as it receives an arterial blood supply and has been shown to exhibitchemoreceptor and baroreceptor activity (Laurent, 1967).

Cardiac output rose approximately 50% as perfusion rate increased from 85 ml/minto the highest rates tested. As mentioned earlier, Fick principle calculations of Q inthis study may be somewhat inaccurate due to a lack of JPQO, data. In addition, theQ calculations may have been affected in perfused fish by 'washing out' of blood COa

owing to the high solubility of COg in water and the persistent gradient between bloodand water at high perfusion rates. It appears that inaccuracies in calculated Q werepresent for the perfused fish as T^ approximately doubled over the perfusion raterange while calculated Q increased by only about 50%. Since arterio-venous oxygendifference did not change appreciably over the flow range (Fig. 5), except at the lowestflow rate, the only means of increasing tr

Ot was by elevation of Q. Thus a doubling


of J'o, should have been accompanied by a doubling of Q. These results show thatFick principle calculations of Q are subject to the same limitations as Fick principlecalculations of V'Q. Reliable Fick Q requires accurate knowledge of arterial and venousPO t and POo, levels and a representative blood curve. Errors in Q in the present studyare probably a result of lack of P(x>, data.

Oxygen uptake increased as perfusion rate went up (Fig. 5) despite the fact thatutilization fell (Fig. 4) and anatomical deadspace in the gills had probably increased(Hughes, 1966). Assuming that our calculations of Q are sufficiently accurate to showlarge changes in Q, the rise in Vo% can be attributed to two factors: (1) an increase incardiac output and (2) a vasodilation of the gills. Cardiac output rose with perfusionrate but there was no elevation of dorsal aortic blood pressure (Fig. 6). In fact, bloodpressure decreased slightly as perfusion rate went up. Since blood pressure did notrise in the presence of elevated Q there must have been a vasodilation of blood vesselsin both the gills and peripheral circulation. Vasodilation of the gills could open addi-tional vascular exchange areas (Steen & Kryusse, 1964; Richards & Fromm, 1969)and lead to a reduction in physiological deadspace (Hughes, 1966). Also, the 50%increase in transfer factor that occurred as flow went up suggests that adjustmentsfavouring the transfer of oxygen across the gills had occurred. An increase in transferfactor could be the result of increased surface area of the gills, a decrease in diffusiondistance for oxygen, an increase in the diffusion coefficient for oxygen or a combinationof these factors (Randall et al. 1967).

The rise in T Oi, Q and vasodilation could result from the fish becoming disturbedor excited at high flows. During excitement catecholamines liberated into the blood(Nakano & Thomlinson, 1966) act on a-adrenergic receptors and may cause dilationof gill blood vessels (Randall & Stevens, 1967). These catecholamines could alsoelevate cardiac output by increasing the rate and force of contraction of the heart(Randall, 1968).

Another possibility is that ventilation and circulation may be controlled so thatcardiac output and Vo are matched. It does not appear that Vo increases in responseto elevated cardiac output alone in trout since Vo does not rise during anaemia despitea tenfold increase in Q (Cameron & Davis, 1970). However, the capacity-rate ratio(Hughes & Shelton, 1962) was maintained in anaemic fish. The present study showedthat Q increased as perfusion rate went up which would result in a tendency tomaintain capacity-rate ratio. This rise in Q, accompanied by a vasodilation of thegills, would facilitate gas exchange at high Po and would meet the increased exchangerequirements associated with activity or excitement.

Utilization of oxygen from the inspired water was exceedingly low at high perfusionrates (Fig. 4). This is a result of the high flow rates and short residence time of thewater in the gills and may also reflect poor exchange conditions at the gills at highflows. When flow rate is high adjacent gill filament tips are drawn apart and a portionof the respiratory flow spills past the tips without contacting the lamellae (Saunders,1961; Pasztor & Kleerekoper, 1962). Hence, a large proportion of the total flow wouldfollow a non-respiratory pathway and low utilization would result. Also, interlamellarflow rate could be high enough to prevent water in the centre of each interlamellarspace from exchanging gases at all (see fig. 3 in Hughes, 1966). Active fish withhigh VQ have more closely spaced lamellae than sluggish fish (Hughes, 1966).


Closely spaced lamellae would reduce interlamellar anatomical deadspace at highflow rates.

It is also possible that interlamellar flow decreases at high flows owing to distortionof the lamellae and abduction of the gill filaments to allow non-respiratory spillage ofwater. The fusion of gill filaments in tuna (Muir & Kendall, 1968, 1969) may reducelamellar distortion and keep the lamellae close together to facilitate utilization athigh flow rates.

Artificial perfusion of the gills by means of a mouth tube appears to provide a lessthan optimum flow pattern over the gills. The perfusion studies do provide usefulinformation on the control of circulation and ventilation and emphasize the importanceof efficient distribution during normal ventilation. In addition they show that gasexchange is not drastically inhibited at high perfusion rates even though anatomicaldeadspace may be high.

The limitations of mouth perfusion should be considered by those contemplatingits use in experiments or surgical procedures. A flared perfusion tube might distributewater more uniformly to the gills. Care should always be taken to keep perfusionrates high to ensure saturation of the arterial blood of perfused fish.

SUMMARY

1. Ventilation volume was measured directly in rainbow trout using a rubbermembrane attached to the mouth which separated inspired and expired water andallowed collection of the latter.

2. Mean ventilation volume at 8-6 °C for 18 trout weighing approximately 200 gwas 37+i-8 ml/min/fish. Mean ventilation rate and ventilatory stroke volumeaveraged 74 breaths/min and 0-5 ml/breath respectively.

3. Ventilation volume could be increased nearly sevenfold during moderate, short-term hypoxia as a result of a large increase in ventilatory stroke volume and a smallincrease in ventilation rate.

4. The ratio between the flow rates of water and blood through the gills wasapproximately 10.

5. Percentage utilization of oxygen from inspired water had a mean of 46± 1*5%and ranged from 23 to 64%.

6. Artificial perfusion of the gills with water at different flow rates was achieved bytying a tube into the mouth of trout.

7. Perfused fish could not saturate their arterial blood with oxygen at a perfusionrate of 45 ml/min but could do so at rates ranging from 85 to 1200 ml/min.

8. Low arterial tensions at a perfusion rate approximating the mean Vo of fish withoral membranes are probably the result of a poor pattern of water flow over the gillsduring perfusion.

9. Opercular movements occurred only at perfusion rates below 700 ml/min andincreased in frequency as perfusion rate dropped. This ventilatory activity may haveresulted from receptors sensitive either to water flow over the gills or to arterial POt.

10. As perfusion rate went up cardiac output and oxygen uptake increased. Thesechanges were accompanied by a drop in dorsal aortic pressure which reflected vaso-dilation of the gills and peripheral circulation. This change in the pattern of blood


flow through the gills contributed to a 50 % increase in oxygen transfer factor acrossthe gills.

11. At the highest perfusion rates there was no apparent impairment of gas exchangeeven though anatomical deadspace was probably high.

We would like to thank Dr D. J. Randall who provided guidance during this studyand assisted greatly in the preparation of this manuscript. The comments of Dr G.Holeton and Mr C. Wood are greatly appreciated. Miss S. Bourque provided tech-nical assistance during the hypoxia experiments. Dr Murray Newman and theVancouver Public Aquarium Association generously provided research space for thisstudy. The work was supported by grants from the National Research Council ofCanada and the British Columbia Heart Foundation and an NIH Postdoctoral Fellow-ship award (1-FO2-HE36334-O1) to J. N. C. from the U.S. National Heart Institute.

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